本文關(guān)鍵詞：丙型肝炎病毒多表位蛋白的抗原性及免疫應(yīng)答研究　出處：《第二軍醫(yī)大學(xué)》2005年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 丙型肝炎病毒(Hepatitis C virus、HCV) 乙肝表面抗原(HBsAg) 模擬表位 多表位 DNA疫苗 融合抗原 免疫應(yīng)答

【摘要】：丙型肝炎病毒(hepatitis C virus,HCV)主要通過(guò)血液途徑傳播,能引起急、慢性肝炎并可導(dǎo)致肝硬化和肝細(xì)胞癌。全世界現(xiàn)有HCV感染者約1.7億,嚴(yán)重影響人類健康,其治療常用干擾素和利巴韋林,應(yīng)答率也僅約為40%,因此,研制HCV疫苗具有重要意義。HCV基因組全長(zhǎng)一般在9379～9481nt之間,編碼一個(gè)約含3000個(gè)氨基酸的多聚蛋白前體,在宿主和病毒自身蛋白酶的加工下產(chǎn)生結(jié)構(gòu)蛋白(C、E1和E2)和非結(jié)構(gòu)蛋白(NS2、NS3、NS4和NS5等)。結(jié)構(gòu)蛋白E2中的第384-410位氨基酸區(qū)段是個(gè)高度變異區(qū),稱為HVR1。眾多實(shí)驗(yàn)表明,HVR1中存在中和表位,其相應(yīng)的中和抗體不僅可以阻止病毒對(duì)靶細(xì)胞的粘附,而且在感染痊愈過(guò)程中起著重要作用,但該區(qū)段高度變異,誘生的抗體具有株特異性,往往對(duì)不同HCV變異株的感染缺乏交叉保護(hù),給丙型肝炎疫苗的研制造成困難。 本實(shí)驗(yàn)室在前期工作中,曾針對(duì)HCV的免疫學(xué)特性,優(yōu)選了數(shù)條能模擬大多數(shù)HVR1免疫原性的HVR1模擬表位,同時(shí)聯(lián)合應(yīng)用多條HCVT細(xì)胞表位,設(shè)計(jì)合成出了一段包含9條HCVE2蛋白HVR1(384～410aa)模擬B細(xì)胞表位,2條C區(qū)保守CTL表位(35～44aa,132～140aa)、1條NS3區(qū)保守CTL表位(1073～1081aa)及1條NS3區(qū)保守Th表位(1251～1259aa)的多表位抗原融合基因(multi-epitope fragment combination,mfc),并對(duì)其免疫原性進(jìn)行了分析,發(fā)現(xiàn)該多表位抗原基因能夠誘生針對(duì)HCV的特異性免疫應(yīng)答。在此基礎(chǔ)上,為進(jìn)一步提高mfc基因的免疫原性,本研究將HAsAg基因融合在mfc基因的N端,構(gòu)建了以HBsAg為佐劑的HCV DNA疫苗,在小鼠中檢測(cè)了其免疫效果;同時(shí),本研究從mfc基因中優(yōu)選出5條HVR1模擬B細(xì)胞表位基因(hcvme),與gst基因融合后經(jīng)大腸桿菌表達(dá)并純化出重組蛋白GST-HCVME,通過(guò)小鼠免疫實(shí)驗(yàn)對(duì)其免疫原性進(jìn)行了分析;此外,本研究還檢測(cè)了mfc基因在大腸桿菌中的重組表達(dá)產(chǎn)物—GST-MFC蛋白對(duì)丙肝患者外周血淋巴細(xì)胞的增殖作用情況。 一、以乙型肝炎表面抗原基因?yàn)樽魟┑谋透窝撞《径啾砦籇NA疫苗的構(gòu)建及在小鼠中的免疫應(yīng)答研究 為增強(qiáng)HCV多表位DNA疫苗的免疫效果,我們將HBsAg基因融合在mfc基因的N末端,構(gòu)建了HBsAg+MFC的重組HCV DNA疫苗,體外轉(zhuǎn)染HEK 293T細(xì)胞檢測(cè)了其瞬時(shí)表達(dá)情況,體內(nèi)實(shí)驗(yàn)采用肌肉注射法接種BALB/c小鼠對(duì)其免疫效果進(jìn)行了評(píng)價(jià),檢測(cè)了其誘導(dǎo)的抗體產(chǎn)生情況、特異性抗體同HVR1合成肽的交叉反應(yīng)率、MFC蛋白特異性的免疫小鼠脾細(xì)胞增殖反應(yīng)及細(xì)胞因子IFN-γ
[Abstract]:Hepatitis C virus hepatitis C virus (HCV) is mainly transmitted through blood channels, can cause acute. Chronic hepatitis can also lead to cirrhosis and hepatocellular carcinoma. There are about 170 million HCV infected people in the world, seriously affecting human health, its commonly used interferon and ribavirin treatment, the response rate is only about 40%. Therefore, the development of HCV vaccine is of great significance. The length of HCV genome is generally between 9379 and 9481 NT, encoding a polyprotein precursor containing about 3000 amino acids. Under the processing of host and virus autoproteinases, structural proteins (Con E1 and E2) and nonstructural proteins (NS2 + NS3) were produced. NS4 and NS5 et al. The 384-410 amino acid region of structural protein E2 is a highly variable region called HVR1.Many experiments indicate that neutralization epitopes exist in HPVR1. The corresponding neutralizing antibody can not only prevent the virus adhesion to target cells, but also play an important role in the healing process of infection. It is difficult to develop hepatitis C vaccine because of the lack of cross protection against infection of different HCV variants. In our previous work, we selected several HVR1 mimic epitopes which can mimic the immunogenicity of HVR1 in view of the immunological characteristics of HCV. At the same time, using multiple HCVT cell epitopes, we designed and synthesized a segment containing 9 HCVE2 protein HVR1O384 (410aa) to mimic B cell epitopes. There were 2 conserved CTL epitopes in region C (35 ~ 44aA ~ (132140aa)). A polyepitope antigen fusion gene of a conserved CTL epitope of NS3 region 1073, 1081aa, and a conserved Th epitope of NS3 region, 1251a (1259aa). Multi-epitope fragment combination. It was found that the multiepitope antigen gene could induce the specific immune response to HCV. On this basis, the immunogenicity of mfc gene could be further improved. In this study, the HAsAg gene was fused into the N-terminal of mfc gene, and the HCV DNA vaccine with HBsAg as adjuvant was constructed, and its immune effect was tested in mice. At the same time, five HVR1 mimic B cell epitope genes (hcvme) were selected from mfc gene. The recombinant protein GST-HCVMEwas expressed and purified by E. coli after fusion with gst gene. The immunogenicity of GST-HCVMEwas analyzed by immunological assay in mice. In addition, GST-MFC protein, a recombinant expression product of mfc gene in Escherichia coli, was used to detect the proliferation of peripheral blood lymphocytes in patients with hepatitis C. 1. Construction of hepatitis C virus multiepitope DNA vaccine with hepatitis B surface antigen as adjuvant and its immune response in mice In order to enhance the immune effect of HCV multiepitope DNA vaccine, we fused HBsAg gene into the N-terminal of mfc gene. The recombinant HCV DNA vaccine of HBsAg MFC was constructed and its transient expression was detected by transfection of HEK 293T cells in vitro. In vivo, the immune effect of BALB/c mice was evaluated by intramuscular injection, and the production of specific antibody and the cross-reaction rate between specific antibody and HVR1 synthetic peptide were detected. Proliferative response of spleen cells and cytokine IFN- 緯 in immunized mice with specific MFC protein
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2005
【分類號(hào)】：R392

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